Influenza viruses are one of the most ubiquitous viruses present in the world, affecting both humans and livestock. Influenza results in an economic burden, morbidity and even mortality, which are significant. Influenza viruses cause epidemics almost every winter, with infection virus rates as high as 40% over a six-week period. Influenza infection results in various disease states, from a sub-clinical infection through mild upper respiratory infection to a severe viral pneumonia. Typical influenza epidemics cause increases in incidence of pneumonia and lower respiratory disease as witnessed by increased rates of hospitalization or mortality.
Vaccination plays a critical role in controlling annual influenza epidemics. Currently available influenza vaccines are either inactivated or live attenuated influenza vaccines.
While the vast majority of current influenza vaccines, for seasonal flu, are non-adjuvanted vaccines, other approaches such as for pandemic vaccines rely on adjuvantation in order to be able to decrease the antigen content (antigen sparing) and thus increase the number of vaccine doses available. The use of an adjuvant may also be useful for overcoming the potential weak immunogenicity of the antigen in specific subjects such as in a naïve or immuno-compromised population. By way of examples, may be cited influenza viruses adjuvanted with aluminium salt, such as aluminium hydroxide or aluminium phosphate, or with oil-in-water emulsion. A sub-unit influenza vaccine adjuvanted with the oil-in-water emulsion MF59 is commercially available.
A classical assay for standardizing antigen content of influenza vaccines is the single radial immunodiffusion (SRID) assay (J. M. Wood et al.: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330), which was recommended by the WHO in 1978 to replace tests based on agglutination of erythrocytes. The assay is based on the immunodiffusion of influenza antigens, such as hemagglutinin (HA) antigen, into an agarose gel containing specific anti-HA serum, during which antigen/antibody complexes will be formed within the gel. HA antigen diffuses radially from the wells and reacts with specific antibodies, producing a zone of opalescence in the gel, typically in the form of a ring. The area of the reaction zone surrounding antigen-containing wells is an estimate of the quantity of antigen added to the well. Upon gel staining, the surface of these rings is measured, and the antigen content of a virus preparation of a certain subtype is calculated by using a calibration curve obtained with an antigen reference batch of this subtype with a known HA content.
However, such an assay may not be suitable for any type of influenza preparations or compositions, in particular, for adjuvanted preparations, as the presence of adjuvant may interfere with the normal test.
Therefore, there is a need to provide improvements to the SRID assay, so as to broaden its applicability and, in particular, to make it suitable for adjuvanted preparations or compositions, such as adjuvanted vaccines.
WO 2009/081172 discloses a modified influenza SRID assay protocol for an adjuvant-adsorbed antigen, in particular, for aluminium salt-adjuvanted vaccines, which includes an additional step in which the antigen is desorbed from the adjuvant prior to diffusion.
Therefore, there is still a need to provide for a modified SRID assay, in particular, suitable for assessing adjuvanted vaccines, which is easy and fast to implement, and applicable for identifying and/or quantifying antigens, such as HA, in any type of virus preparation, whether adjuvanted or not, including final vaccine formulations.